Hypoxic cells in solid tumors are known to resist damage by ionizing radiation and some chemotherapeutic agents, yet hypoxic fraction is not routinely monitored in human tumors largely because methods are still inadequate or impractical. The overall aim of this proposal continues to be the development of methods to identify and quantify hypoxic cells in solid tumors. During the current funding period, a method developed in this laboratory for detection of hypoxic cells in solid tumors will be evaluated in murine and human tumors. Based on single cell microelectrophoresis, the 'comet' assay is used to quantify DNA strand breaks in individual cells. Radiobiologically hypoxic cells are about three times more resistant to DNA damage than aerobic cells, thus providing the basis for measuring hypoxic fraction in single cell suspensions prepared from solid tumors. There are several advantages to this approach which has been shown to quantify hypoxic fraction in SCCVII murine tumors. It is now necessary to determine the general applicability of the assay. The first Specific Aim is therefore to apply the comet assay to a panel of murine tumors chosen to exhibit a range of hypoxic fractions as well as different types of hypoxia (i.e., acute versus chronic hypoxia). Hypoxic fraction will be measured in Lewis lung, WiDr, SiHa, KHT and RIF-1 tumors growing in mice, and results compared with a standard method for determining hypoxic fraction, paired- survival curve analysis. Using the SCCVII tumor, hypoxic fraction will be measured in tumor cells versus macrophages, in cells close to versus distant from the blood supply, after high (5-15 Gy) and low (2 Gy) doses of radiation, and in proliferating versus non-proliferating tumor cells. In a second Specific Aim, hypoxic fraction in 10-20 accessible human tumors will be measured in tumor cells recovered from fine needle aspirates. Since reoxygenation rate may be more critical for successful radiotherapy than pre-treatment hypoxic fraction, a third Specific Aim is directed towards the development and application of a fluorescence image analysis method for quantifying changes in tumor perfusion at different times following exposure of SCCVII tumors to ionizing radiation. Results will be compared with oxygen consumption by tumor cubes, cell recovery, and hypoxic fraction measured using the comet assay. The comet assay will hopefully provide a practical method to assess both pre- and post-treatment hypoxic fractions in solid human tumors, and together with ongoing studies of tumor perfusion, tumor oxygen consumption and vascular density, will help to determine the role of hypoxia in the response of tumors to ionizing radiation.